Biology subjects

Harkness, R. W.

Publications and source records attributed to Harkness, R. W..

4 recordsLinked to original sources

bioRxiv2026

Navigating the oligomeric landscape of the periplasmic stress response protease-chaperone DegP with charge detection mass spectrometry

DegP is a periplasmic protease-chaperone essential for protein quality control and virulence factor trafficking in Gram-negative bacteria. In its apo form, DegP adopts a dynamic ensemble of oligomers derived from trimer building blocks through two competing self-assembly pathways. Upon engaging client proteins, apo DegP oligomers redistribute into discrete cage structures inside which the clients are encapsulated. The cage ensembles formed depend on the size of the bound clients, and notably can include 12mers, 24mers, and 60mers. Previous studies mapped the DegP oligmeric landscape using dynamic light scattering, analytical ultracentrifugation, nuclear magnetic resonance spectroscopy, and electron cryomicroscopy, modalities which in general report ensemble averages and often cannot directly delineate closely related coexisting species. Here, we apply charge detection mass spectrometry (CDMS) to directly measure the masses of individual DegP ions, resolving the complete oligomeric distribution in the absence and presence of four clients of increasing size. We reveal previously undetected odd-numbered oligomers and quantify the relative abundance of each assembly. Through heat-cool cycling CDMS experiments, we track cage distribution changes and reveal client protection and refolding, providing a direct view of the chaperone capabilities of DegP. These results further establish CDMS as a powerful single-molecule tool for dissecting heterogeneous protein assembly landscapes.

biochemistry↗

bioRxiv2026

Competing effects modulate the rate of poly(A) RNA deadenylation in a biomolecular condensate

The unique solvent milieu found in biomolecular condensates can control cellular enzymatic reactions and shift reaction kinetics by modulating reactant concentrations, structural dynamics, and enzyme activities. Here we explore the interplay of multiple regulatory factors within a condensate to control poly(A) RNA deadenylation, the first and rate-limiting step in mRNA turnover. The deadenylase CNOT7, a subunit of the CCR4-NOT deadenylation complex, localizes to cytoplasmic RNA granules and shows increased degradation activity in vitro in condensates formed by the C-terminal low complexity disordered region of CAPRIN1, a component of RNA granules. We use a combination of enzymatic assays, kinetic modeling, microscopy, Nuclear Magnetic Resonance (NMR) spectroscopy, and molecular dynamics simulations to deconvolute and define the components that underlie this enhancement. We found that enzyme and RNA are concentrated in condensates relative to buffer, which increases CNOT7 activity, while the equilibrium between CNOT7s active and inactive states remains unchanged. The concentration-dependent increase in enzymatic rates is counterbalanced by a substantial decrease in the enzymes catalytic efficiency, likely due to slower diffusion of CNOT7 and RNA within the condensates, which lessens the probability of enzyme-substrate complex formation. Molecular dynamics simulations reveal CNOT7-CAPRIN1 interactions that rely on conserved CAPRIN1 sequence features, hinting at an evolutionarily conserved role for CAPRIN1 condensation. With this quantitative kinetic analysis, we describe the multifaceted mechanism behind regulation of CNOT7 deadenylation by a condensate environment. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/736149v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@51cd47org.highwire.dtl.DTLVardef@7bfccaorg.highwire.dtl.DTLVardef@114375org.highwire.dtl.DTLVardef@163f4ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Mechanism of allosteric activation in human mitochondrial ClpP protease

Human ClpP protease contributes to mitochondrial protein quality control by degrading misfolded proteins. ClpP is overexpressed in cancers such as acute myeloid leukemia (AML), where its inhibition leads to the accumulation of damaged respiratory chain subunits and cell death. Conversely, hyperactivating ClpP with small-molecule activators, such as the recently-discovered ONC201, disrupts mitochondrial protein degradation and impairs respiration in cancer cells. Despite its critical role in human health, the mechanism underlying the structural and functional properties of human ClpP remain elusive. Notably, human ClpP is paradoxically activated by active-site inhibitors. All available structures of human ClpP published to date are in the inactive compact or compressed states, surprisingly even when ClpP is bound to an activator molecule such as ONC201. Here, we present the first structures of human mitochondrial ClpP in the active extended state, including a pair of structures where ClpP is bound to an active-site inhibitor. We demonstrate that amino acid substitutions in the handle region (A192E and E196R) recreate a conserved salt bridge found in bacterial ClpP, stabilizing the extended active state and significantly enhancing ClpP activity. We elucidate the ClpP activation mechanism, highlighting a hormetic effect where sub-stoichiometric inhibitor binding triggers an allosteric transition that drives ClpP into its active extended state. Our findings link the conformational dynamics of ClpP to its catalytic function and provide high-resolution structures for the rational design of potent and specific ClpP inhibitors, with implications for targeting AML and other disorders with ClpP involvement. Significance statementHuman ClpP protease is essential for maintaining mitochondrial protein quality by degrading damaged proteins. In cancers like acute myeloid leukemia (AML), ClpP is overexpressed, and inhibiting it causes cancer cell death by disrupting mitochondrial function. Conversely, activating ClpP with small molecules, such as ONC201, also leads to cancer cell death by impairing mitochondrial respiration. However, the structural details of ClpP activation have been elusive. Our research presents the first structures of human ClpP in its active state, revealing a novel activation mechanism where inhibitors unexpectedly trigger activity through allosteric changes. These insights provide a foundation for designing targeted therapies for AML and other diseases where ClpP plays a crucial role.

biochemistry↗

bioRxiv2022

Flexible client-dependent cages in the assembly landscape of the periplasmic protease-chaperone DegP

The periplasmic protein DegP, that is implicated in virulence factor transport leading to pathogenicity, is a bi-functional protease and chaperone that maintains protein homeostasis in gram-negative bacteria. To perform these functions, DegP captures clients inside cage-like structures, which we have recently shown to form through the reorganization of high-order preformed apo-oligomers, consisting of trimeric building blocks, that are structurally distinct from client-bound cages. Our previous studies suggested that these apo oligomers may allow DegP to encapsulate clients of various sizes under protein folding stresses by forming cage ensembles that can include extremely large cage particles. To explore the relation between cage and substrate sizes, we engineered a series of DegP clients of increasing hydrodynamic radii and analyzed their influence on DegP cage formation. We used dynamic light scattering and cryogenic electron microscopy to characterize the hydrodynamic properties and structures of the DegP cages that are adopted in response to each client. We present a series of flexible cage structures including novel 30mer and 60mer particles. Key interactions between DegP trimers and the bound clients that stabilize the cage assemblies and prime the clients for catalysis are revealed. We also provide evidence that DegP can form cages which approach subcellular organelles in terms of size. Significance statementGram-negative pathogens export virulence factors that interfere with the function of host cells. This process is mediated by DegP, a protein which controls protein homeostasis in the periplasm of these bacteria and thus is a target for the development of novel antibiotics. DegP operates by incorporating client proteins inside cage-like structures to either recycle them or protect them from aggregation. Using a combination of dynamic light scattering measurements and cryogenic electron microscopy, we have shown that DegP can adopt many types of cages, some as large as subcellular organelles, depending on the size of the engaged client. This property likely enables DegP to capture different sized clients in response to protein misfolding stresses.

biophysics↗